Richard L. Moldwin

Establishment and characterization of a megakaryoblast cell line with amplification of MLL

A new cell line with megakaryoblastic features, designated UoC-M1, was established from the malignant cells of a 68-year-old patient with acute myeloid leukemia. The patient’s leukemic cells reacted with α-naphthyl acetate esterase and acid phosphatase and expressed CD7, CD24, CD34, CD38, CD45, HLA-DR and CD61. Cytogenetic analysis of the patient’s malignant cells (and of the UoC-M1 cells) showed a human, male hypodiploid karyotype with many chromosome rearrangements and marker chromosomes. Spectral karyotyping (SKY) analysis complemented the G-banded karyotyping and clarified several chromosomal translocations and identified the marker chromosomes. Fluorescence in situ hybridization (FISH) and SKY analysis demonstrated that one marker chromosome contained three segments of chromosome 9 interspersed with three segments of chromosome 11, as well as a portion of chromosome 19. FISH analysis with a probe for MLL revealed that the UoC-M1 cells contained four copies of the MLL gene. Southern blot analysis determined that the MLL gene had a germline profile while Northern and Western analyses showed that the MLL mRNAs and protein were of the appropriate sizes. This is the first report of amplification of the MLL gene which may be an additional mechanism of leukemogenesis or disease progression.

Obtaining an accepted Investigational New Drug application to operate an umbilical cord blood bank.

BACKGROUND: The residual blood left in the placenta, previously considered a biologic waste, contains sufficient hematopoietic stem and progenitor cells to consistently engraft at least a small recipient. Over the past several years, more than 500 HLA‐matched, related and unrelated, allogeneic cord blood transplants have been performed. Consequently, public and private cord blood banks are being developed to meet future demands. Thus, the definition of a suitable and effective cord blood component needs to be critically defined. In February 1997, the US Food and Drug Administration (FDA) proposed that cord blood banks should operate under an Investigational New Drug (IND) license.

Enhanced effect of vascular endothelial growth factor, thrombopoietin peptide agonist, SCF, and Flt3-L on LTC-IC and reporter gene transduction from umbilical cord blood CD34+ cells.

BACKGROUND: Hemangioblastic precursors have been identified that give rise to both endothelial cells and HPCs, suggesting that common growth factor requirements may exist.

Interleukin-2 differentially regulates IL-2 receptors on murine cloned cytolytic and helper T cells

The effects of interleukin-2 (IL-2) on the expression of IL-2 receptors by cloned cytolytic and helper T lymphocytes were studied using three anti-IL-2 receptor antibodies. IL-2 enhanced the expression of IL-2 receptors on all clones tested. A steep dose-response curve was observed with no measurable effect seen below 2 units/ml and maximal IL-2 receptor expression with greater than 5 units/ml. IL-2 receptor expression peaked 24-48 hr after the addition of IL-2. The subsequent decrease in IL-2 receptor expression correlated with a decrease in the levels of IL-2 remaining in the culture supernatants of cytolytic T lymphocyte cells. Removing residual IL-2 from cultures resulted in the rapid return of IL-2 receptor expression to unstimulated levels. The daily addition of low levels of IL-2 to cultures resulted in the prolonged expression of high levels of IL-2 receptors by non-IL-2-producing cloned cytolytic T cells. Cloned helper T cells which make IL-2 showed the initial increase in IL-2 receptor levels, but the daily addition of IL-2 did not prolong IL-2 receptor expression in these cells. These data suggest that IL-2 receptors on those cells which do not make IL-2 are regulated differently from receptors on cells which themselves make IL-2.

Expansion of CD34+kdr+ cells in cord blood after culture with tpo, FLT-3l, scf, and vegf

Abstract CD34+ cells that co-express KDR (a VEGF receptor) have been identified in umbilical cord blood (CB) and shown to be rich in primitive stem cell activity. We studied the production of CD34+KDR+ cells in stem cell “expansion” cultures. CD34+ cells were isolated from CB and cultured for 2, 4, and 6 weeks in serum-free medium supplemented with 5% CB plasma in the presence of rhSCF (20 ng/ml), rhTPO, rhFLT-3L and ± rhVEGF (50 ng/ml). By flow cytometry, the fraction of CD34-selected cells that were also KDR+ was 1.6 ± 0.3% prior to culture. After 2 weeks in culture, in the presence of rhSCF, rhTPO, and rhFLT-3L, the percent of CD34+KDR+ cells increased to 5.7 ± 3.2% with a CD34+KDR+ increase of 112-fold and all overall increase of 43-fold in total cell number. After 4 weeks in culture (± VEGF), CD34+KDR+ cells had increased >200-fold. Total cell expansion at week 6 was: 121-fold and 110-fold (with or without VEGF added, respectively). VEGF supplemented cultures had slightly greater numbers of CFU-GM and HPP-CFC. In addition, after 2 weeks in culture (without VEGF), we observed that 7–26% of the KDR+CD34+ were CD38− while cultures with VEGF contained 13–38% CD34+KDR+CD38− cells, possibly indicating the presence of a more primitive stem cell subset in these cultures.

Growth inhibition of B-cell precursor acute lymphoblastic leukemia cell lines by monocytes: A role for prostaglandin E2

Leukemic cell lines have proven invaluable in the molecular analysis of recurring chromosomal translocations but the optimal methods for leukemia cell line establishment are unknown. During in vitro culture, most B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells die within 1 week at least partially mediated by inhibitors elaborated by peripheral blood mononuclear cells (PB MNCs) present within the leukemia sample. In experiments reported here, cyclooxygenase inhibitors (indomethacin and meclofenamic acid) blocked the PB MNC-mediated inhibition of BCP-ALL proliferation. Also, prostaglandin E2 (PGE2) was detected in supernatants from PB MNC cultures. When PGE2 was mixed directly with BCP-ALL cells, proliferation decreased significantly. Under the culture conditions used, PB MNCs secreted PGE2 which appears to be one of the major inhibitors of BCP-ALL growth in vitro.

Regulation of Activation of Cloned Murine T Cells

Although helper T lymphocytes (HTL) and cytolytic T lymphocytes (CTL) share a number of common features, several lines of evidence indicate that they are controlled by distinctive regulatory mechanisms. Specifically, we have found that cloned murine CTL, in addition to proliferating in response to IL-2, also have an IL-2-independent pathway for replication; however, an IL-2-independent pathway has not been identified in our cloned murine HTL. HTL which secrete IL-2 become unresponsive to antigen following exposure to IL-2 while CTL do not; the limb of the T cell activation pathway leading to an increase in intracellular calcium appears to be selectively affected in this unresponsive state. In CTL–HTL hybrids constructed using a drug-marked variant of a cloned murine CTL, secretion of the full array of lymphokines can be induced by stimulating the antigen receptor of either the CTL or HTL partner. However, only target cells bearing antigens with which the CTL partner reacts can be lysed by the CTL–HTL hybrid cells; target cells bearing antigens with which the HTL partner reacts cannot be lysed. These differences appear to reflect separate regulatory mechanisms that control the responses of functionally distinct T cell subsets.